Project Abstract: This application is being submitted to PA-18-591 as a supplement to our current award R01AG051086. We seek to develop the resource beyond its core goal by testing miRNAs in the Induced Pluripotent Stem cells (also known as iPS cells or iPSCs) generated directly from adult cells of subjects with Alzheimer's disease (AD) or controls. AD is the most common cause of dementia in the elderly. Because current treatments provide modest symptomatic relief and do not slow AD progression, a better understanding of molecular bases of AD pathology is needed. This proposal will identify and validate microRNA (miRNA), a new class of drug targets. miRNA are endogenous, short, non-coding RNAs that generally inhibit protein expression by interacting with specific recognition elements of target transcripts. AD is believed to result from overproduction of amyloid-? peptide (A?), derived from A? precursor protein (APP), and dysregulation of proteins involved in A? production (e.g. APP, ?-secretase/BACE1) contributes to excess A? deposition. Therefore, we propose to study APP and BACE1 regulation using miRNA strategies. We hypothesize that specific miRNAs regulate endogenous levels of APP and BACE1, are dysfunctional in AD, and manipulation of these miRNAs will reduce A?. Specific Aim 1(SA1) will identify functional miRNA target sites in APP and BACE1 and validate miRNA post- transcriptional regulation of native APP and BACE1 expression. Rationale: Discover functional miRNA targets in UTRs of APP and BACE1 transcripts using iPS neuron cultures. Endpoints are APP and BACE1 mRNA & proteins, and A? peptides, which we predict to change with miRNA. Impact: Manipulation of miRNA regulation is a novel therapeutic approach and may be feasible for correcting gene dysregulation in AD. SA2 will test physiological interactions between miRNA validated in SA1 and their regulatory networks over APP and BACE1 expression. Rationale: Test other mechanisms of regulation that are likely linked to maintain homeostasis of APP and BACE1, and A?. Identifying roles of miRNA in this network is vital to pharmacologi- cally target miRNA-transcript interactions. Impact: Reveal novel mechanisms for miRNA function in AD. The major advantage of the proposal is that appropriate iPS cultures can be had from individuals who have i) autosomally dominant (familial) AD, ii) sporadic (apolipoprotein E ?4, associated) AD, and iii) age-matched non-AD controls. Comparison of effects from all three donor backgrounds would specifically elucidate differ- ences among specific rare and relatively more common forms of AD and both vs. normal individuals. All experi- ments in this proposed supplement fall fully within the scope of the original R01 grant application. Overall, the proposed novel research dictated by our central hypothesis is likely to have a significant para- digm shift on the field by elucidating novel regulatory mechanisms and identifying how specific miRNAs regu- late important gene products implicated in AD. The final impact will be in eventual use of these new drug tar- gets to produce therapeutic agents to slow or halt disease progression in AD.